Malaria is affecting 40% of the world's population with an estimated 1.5-2.7 million deaths annually (57). This represents a tremendous human suffering and a burden that prevents the development of the affected endemic communities. Malaria is now almost confined to the poorest tropical areas of Africa, Asia and Latin America, but transmission is being reintroduced to areas where it had previously been eradicated. Malaria is one of the world's greatest public health problems.
The increasing emerging of insecticide resistant vectors and drug resistant parasites calls for investment in new and better control tools. Malaria vaccines hold the potential to dramatically alleviate the burden of malaria. However, our understanding of the mechanisms underlying protective immunity is incomplete hence specific markers of protection still needs to be defined.
An effective malaria vaccine will require the induction of appropriate humoral and cellular immune responses, against several key parasite antigens expressed during the various stages of the parasite life cycle. Each stage in the life cycle provides an opportunity for a vaccine.
Two lines of evidence suggest that a malaria vaccine is attainable:
Firstly, it is a well-established observation that repeated exposure to malaria parasites can lead to the development of solid clinical immunity, a status of premunition with concomitant existence of parasites and protective antibodies. Clinically immune individuals generally have a lower parasite density and the immunity is quite effective at reducing mortality.
Secondly, experiments in humans as well as in animal models have established that immunizations can induce immunity against subsequent challenge with parasites suggesting that vaccination can become a realistic tool for malaria control.
In now classical experiments, Cohen and colleagues demonstrated that the passive transfer of antibodies purified from clinically immune individuals could ameliorate acute malaria attacks in African children with life-threatening P. falciparum infections (10). Druilhe and coworkers confirmed Cohen's results (42). They showed that IgG from clinically malaria immune West Africans were able—in a strain-independent manner—to substantially decrease the parasite load in asymptomatic Thai children with drug resistant P. falciparum malaria.
These groundbreaking passive transfer experiments have proven that antibodies are crucial in reducing/eliminating the asexual stage parasite load.
However, in vitro investigations with the same “protective” IgG preparations (42) demonstrated that antibodies do not inhibit parasite growth on their own, but act synergistically with blood mononuclear cells to control parasite multiplication (5). This parasite containing mechanism is referred to as antibody-dependent cellular inhibition (ADCI) (5, 26, 31). Recent studies have further demonstrated that binding of cytophilic antibodies such as IgG1 and IgG3 in conjunction with blood mononuclear cells via their FcγIIa receptors trigger the release of killing factors such as tumor necrosis factor-α (6).
Immuno-epidemiological studies support the in vivo relevance of a monocyte-dependent, antibody-mediated mechanism by showing a correlation between the acquisition of clinical immunity and levels of IgG1 and IgG3 antibodies, which bind well to the monocyte FcγRIIa receptor (1, 41). The putative involvement of this receptor in the development of immunity against clinical malaria is also supported by the finding that allelic polymorphism in FcγRIIa is associated with differential susceptibility to P. falciparum malaria (45). Kenyan infants homozygous for the FcγRIIa-Arg131 allele are reported to be less at risk from high-density P. falciparum infections compared with children with the heterozygous Arg/His131 genotype (45). Since the FcγIIa-Arg131 genotype (but not the FcγIIa-His131 genotype) binds strongly to IgG1 and IgG3, this finding supports the notion that monocyte-mediated killing of P. falciparum is an important mechanism for parasite containment in vivo. Additionally, Aucan et al (2) found that levels of specific IgG2 antibodies—but not IgG3 and IgG1—were associated with protection from clinical malaria in a population from Bukina Faso. Subsequent sequencing of FcγRIIa revealed that 70% of the study subjects had the FcγRIIa-H131 allele. This allele binds strongly to IgG2 (56), suggesting that IgG2 is acting as a cytophilic subclass in this population (2). Collectively these observations suggest that the FcγRIIa genotype is an important factor for the development of immunity to clinical malaria and lends support to the validity of in vitro ADCI model.
The development of a vaccine for malaria has become increasingly recognized as a high priority in the effort to control malaria worldwide due to the increasing incidence of drug-resistant disease. New tools are therefore required to facilitate the clinical evaluation of candidate vaccines, particular the validation of in vitro correlates of the protection afforded by vaccination. ADCI may provide one such tool (13). The currently most prominent blood-stage vaccine candidates MSP1, MSP2, AMA1, and RESA have primarily been selected for clinical testing because of their ability to induce growth-inhibitory antibodies in pre-clinical animal models (9, 16, 16, 30, 55). However, despite initial promises, they have in general proved poorly immunogenic in the human volunteers (18, 25, 29, 43) and the induced antibodies were unable to inhibit the in vitro growth of P. falciparum. Thus, the in vitro invasion inhibition assay is not ready to serve as a surrogate marker of immunity.
The lack of suitable correlates of human protection that reflect inhibition of merozoite invasion has encouraged the development of other in vitro models that reflect possible killing mechanisms in clinically immune individuals. Druilhe and coworkers have hypothesized that antibodies act synergistically with human blood monocytes to control parasite growth in vivo and have accordingly developed the in vitro correlate of this killing mechanism—the ADCI assay. We have so far identified two antigens—GLURP and MSP3—that are targets of ADCI-effective human antibodies.
The Plasmodium falciparum Glutamate-rich protein (GLURP) and the Merozoite surface protein 3 (MSP3) are both targeted by human IgG antibodies, which can inhibit parasite growth in vitro in a monocyte-dependent manner (36, 52) and in vivo in the humanized SCID mouse model (3). The similar effects of human antibodies against these antigens are also suggested by a number of immuno-epidemiological studies, which demonstrate that the levels of GLURP and MSP3 specific cytophilic antibodies (IgG1 and IgG3) are significantly associated with a reduced risk of malaria attacks (11, 38, 50).
The discovery of GLURP and MSP3 is based on the in vitro analysis of passive transfer of immunity by purified African Immunoglobulin G (5, 6, 14, 42). These investigations have led to the elucidation of a putative effector mechanism in the defense against P. falciparum malaria (12), and the subsequent identification of the involved parasite molecules. The major B-cell epitopes recognized by these human IgG antibodies have been localized to conserved sequences in the GLURP27-489 and MSP3212-257 regions, respectively (36, 50, 51). These studies lead to the identification of the N-terminal region of GLURP (GLURP27-489) (52) and the C-terminal region of MSP3, (MSP3210-380) (36) as targets of biologically active antibodies.
Different regions of these antigens have previously been produced in Escherichia coli fused to various affinity-tags (35, 53, 54). Whereas such additional sequences are advantageous for purification they also pose a potential problem because host immune responses against such sequences may render them useless for repeated applications.
Immune epidemiological investigations confirmed the relevance of anti-GLURP and anti-MSP3 IgG antibodies to acquired protection:
For GLURP, three independent studies performed in Dielmo, Senegal (38), Dodowa, Ghana (11, 50) and OoDo, Myanmar (Soe Soe, unpublished) have demonstrated a statistically significant correlation between levels of GLURP-specific IgG3 and/or IgG1 antibodies and protection against malaria attack. This association was highly significant even after controlling for the confounding effect of age-related exposure to P. falciparum. These results confirm previous studies, which found that naturally occurring IgG antibodies to GLURP are associated with protection against disease in Gambian children (15) and against high levels of parasitemia in children from Liberia (21) and Burkina Faso (4).
For MSP3, a high ratio (>2) of cytophilic to non-cytophilic antibodies (IgG1+IgG3/IgG2+IgG4+IgM) allowed to distinguish individuals without recorded malaria attacks from individuals with malaria attacks. This was found in every age group among approximately 200 villagers from Dielmo who have been under daily clinical surveillance for more then 8 years (37). At the individual level, the occurrence of anti-MSP3 IgG3 antibodies was strongly associated with protection, in contrast to antibodies of other isotypes directed against the same molecule or antibodies of any isotype directed against 5 other antigens (37).
A similar consistency in seroepidemiological data is not common for any other malaria vaccine candidate as exemplified by MSP1, the hitherto leading candidate as a vaccine against P. falciparum malaria.
The major B-ell epitopes recognized by these human IgG antibodies have been localized to conserved sequences in the GLURP27-489 and MSP3212-257 regions, respectively (36, 50, 51). These studies lead to the identification of the N-terminal region of GLURP (GLURP27-489) (52) and the C-terminal region of MSP3, (MSP3210-380) (36) as targets of biologically active antibodies.
Sequence analyses of the GLURP27-489 and MSP3210-380 regions from 44 field isolates and laboratory lines of P. falciparum show that defined epitopes in GLURP (P1, P3, and P4) (48) and MSP3 (b peptide) (34), which are targeted by ADCI-effective human antibodies are almost completely conserved, suggesting that they are functionally constrained and not subject to selection for variation at the amino acid level. Of the different epitopes in the GLURP27-489 region, P3 might be the most important, since affinity-purified human antibodies against the P3 peptide mediated the strongest ADCI-effect in vitro (51). The conservation of major B-cell epitopes in GLURP and MSP3 is further supported by the observation that they are almost identical between P. falciparum and the closely related parasite Plasmodium reichenowi; a natural parasite for Chimpanzees (39, 53), and that plasma IgG antibodies from 71 adult Liberians clinically immune to malaria display identical binding patterns towards recombinant proteins representing the GLURP27-500 regions from both species (53).
Collectively, these findings demonstrate that GLURP and MSP3 B-cell epitopes recognized by biologically effective human antibodies are conserved between geographically distant P. falciparum isolates and functionally constrained, suggesting that a vaccine based on GLURP and MSP3 may protect against a broad range of parasite strains worldwide.
In vitro experiments showed that naturally occurring affinity-purified human antibodies to GLURP (52) and MSP3 (36) could inhibit parasite growth in a monocyte-dependent manner, whereas control antibodies affinity-purified on 7 other malarial vaccine candidates were unable to exert a similar effect (47).
The same inhibitory effect was obtained using naturally occurring affinity-purified IgG antibodies against both recombinant proteins (GLURP27-489, and GLURP705-1178) (52) and synthetic peptides derived from the GLURP R0 region, P3 (GLURP93-207), S3 (GLURP407-434), and LR67 (GLURP85-312) (50, 51), respectively.
In vivo experiments where affinity-purified MSP3b-specific human antibodies were passively transferred into P. falciparum infected Hu-RBC BXN mice, showed a parasite clearance as fast as that induced by Chloroquine, and faster than that induced by total African IgG (3). The latter observation indicates that immunization with selected antigens may lead to stronger immunity than that induced by the whole parasite (3).
In vivo experiments where Aotus monkeys immunized with recombinant MSP3 in Freunds complete adjuvant were fully protected against an experimental P. falciparum challenge (20). Immunizations of Saimiri sciureus monkeys have demonstrated that GLURP27-500 adsorbed to Al(OH)3 is non-toxic, immunogenic and elicit high titers of anti-GLURP antibodies which recognize P. falciparum by IFA (8). In a subsequent challenge with P. falciparum infected erythrocytes, two out of three monkeys were partially protected, this effect being directly related to the titer and epitope specificity of the antibodies developed by the primates in response to the immunogen (8).
These findings strongly support the notion that immune responses against GLURP and MSP3 B-cell epitopes that elicit ADCI-effective antibodies controls parasite multiplication in vivo.
Different regions of these antigens have previously been produced in Escherichia coli fused to various affinity-tags (35, 53, 54). Whereas such additional sequences are advantageous for purification they also pose a potential problem because host immune responses against such sequences may render them useless for repeated applications. It is therefore desirable to explore expression systems, which aims to produce the recombinant protein without a vector-encoded affinity-tag.
A restricted number of formulations based on MSP3 and GLURP have been select for further vaccine development and studied at the pre-clinical level first in mice (49, 54) and then in non-human primates challenged with P. falciparum (8). The N-terminal region of GLURP and the C-terminal region of MSP3 proved strongly immunogenic in pre-clinical models. These have now been produced individually using a new, highly efficient, expression system based on the pH and growth phase regulated promoter, P170, from Lactococcus lactis (23, 33).
We have so far identified two antigens—GLURP and MSP3—that are targets of ADCI-effective human antibodies and recently performed two clinical phase I trials with the individual antigens. Both vaccines induced strong cellular responses in the volunteers, whereas the IgG antibody responses were moderate. All volunteers from the GLURP trial generated antibodies against the P3 B-cell epitopes, which is the most prominent target of ADCI-effective antibodies in clinically immune individuals. The relatively low levels of vaccine-induced antibodies may be related to the limited number of B-cell epitopes on the GLURP synthetic peptides.
It is therefore, desirable to develop a vaccine based on a recombinant protein, which include GLURP and MSP3 preferably with neighboring sequences containing additional B- and T-cell epitopes or other antigens from P. falciparum such as the CS-antigen. It is also desirable to use expression systems, which produces the recombinant protein without a vector-encoded affinity-tag, such as L. lactis. 